JP5191036B2 - Anti-human tenascin monoclonal antibody - Google Patents

Description

  The present invention relates to anti-human tenascin monoclonal antibodies, methods and materials for obtaining them, use of the antibodies in the diagnosis and treatment of tumors expressing tenascin, and materials containing the antibodies suitable for use in the medical field.

BACKGROUND OF THE INVENTION The specificity of tumor treatment is often a limiting factor in determining treatment success. In fact, the onset of toxic effects and their poor tolerance by numerous anticancer drugs has limited their use and the quality of life of patients.

  Toxicity reduction is directly related to treatment selectivity for cancer cells only. Monoclonal antibodies are an ideal means for localizing specifically in tumors and, when combined with an avidin / biotin amplification system, constitute a very powerful method of delivering active molecules to tumor sites.

  Tenascin is an extracellular matrix molecule that is expressed during embryogenesis and in adult tissues during the process of scar formation and tumor development, as well as newly formed blood vessels. Tenascin is virtually absent in normal adult tissue, whereas it is expressed in the stroma of many solid tumors, for example: glioma (Burdon, et al., Cancer Res., 43: 2796- 2805, 1983), breast cancer (Chiquet-Ehrismann, et al., 1986), lung cancer (Natali, et al., Intl. J. Cancer, 54: 56-68, 1989), fibrosarcoma and squamous cell carcinoma (Ramos, DM et al., Intl. J. Cancer, 75: 680-687, 1998). Tenascin is expressed in glioma but not in the corresponding normal brain tissue. For a detailed discussion of tenascin, see WO 92/04464, Wistar and related references.

  Based on EP 0 496 074, G. Paganelli et al has developed a method called “three-stage pre-targeting” for systemic and local treatment of tumors, the results of which are reported below Has been: Cremonesi, M., et al., Eur. J. Nucl. Med., 26 (2): 110-120, 1999; Paganelli, G., et al., Eur. J. Nucl. Med. , 26 (4): 348-357, 1999; Paganelli, G., et al., Cancer Biother. & Radiopharm., 16 (3): 227-235, 2001; Grana, C., et al., Br. J. Cancer, 86: 207-212, 2001.

  Other references for this type of cancer treatment are WO 94/04702 and US 5578287.

The three-stage pretargeting therapy, also known as the trademark PAGRIT®, is based on sequential intravenous administration of biotinylated anti-tenascin monoclonal antibody, streptavidin and ⁹⁰ Y-biotin, respectively, streptavidin and ⁹⁰ Y- Avidin and biotinylated albumin are administered prior to biotin (a “chase” step) to reduce the blood levels of antibody and streptavidin. The selectivity of the three-step pretargeting method depends on the use of anti-tenascin monoclonal antibodies. Compared to targeting towards cell surface antigens, targeting of extracellular matrix molecules provides the advantage that they are not affected by modulation of antigen expression by the tumor cells themselves. Pretargeted treatment doses, administration times, and “follow-up” have been established to obtain optimal tumor-to-non-tumor biodistribution ratios.

  Glioblastoma (GBM) or undifferentiated astrocytes subjected to research by Paganelli (Paganelli, G., et al., Eur. J. Nucl. Med., 26 (4): 348-357, 1999) The results obtained in 48 patients suffering from tumors (AA) showed that, except for many examples of allergic reactions to streptavidin, there was virtually no toxicity and the preliminary efficacy of treatment. It was to show. In fact, 2 months after the end of treatment, 25% of patients showed a reduction in mass (complete response = 6%, partial response = 11%, low response = 8%), and 52% of patients remained stable The total response rate was 77%. In many such patients with a life expectancy of less than 6 months, the response to treatment persisted for over a year.

The role of the biotinylated anti-tenascin antibody is to mediate tumor localization and accumulation of avidin and then ⁹⁰ Y-biotin via the biotinylated molecule, directing the radioisotope directly into the tumor. Anti-tenascin antibodies are already the subject of the following patents and patent applications: US 5,624,659, Duke University; JP 2219590, Rikagaku; WO 92/04464, Wistar; and WO 03/072608, Sigma-Tau.

  One specific anti-tenascin antibody is: Siri, A., et al, Nucl. Acid Res., 19 (3): 525-531, 1991; Balza, E., et al, FEBS 332: 39-43, 1993 Its use for therapeutic purposes is: Riva, P., et al., Acta Oncol. 38 (3): 351-9, 1999; Riva, P., et al, Cancer, 80 (12 ): 2733-42; 1997; Riva, P., et al ,. Int. J. Cancer, 5: 7-13, 1992.

  The clone used for the production of the antibody in the above study is called BC-2. The applicant has shown that clone BC-2 is not suitable for industrial development. This is because clone BC-2 produces an additional non-functional light chain (probably from the parental myeloma strain) whose expression levels increase during the development of large-scale production processes, This is because the purification on the scale is hindered.

  Therefore, there is a recognized need for anti-tenascin monoclonal antibodies that can be produced on an industrial scale at the purity level required for pharmaceutical use.

  Previous patent application WO 03/072608, filed by Sigma Tau Industrie Farmaceutiche Riunite SpA, describes the creation of anti-tenascin antibody ST2146 that does not produce an additional non-functional light chain, which is non-functional It can recognize antigenic epitopes shared with antibody BC-4 contaminated with light chains.

  Antibody ST2485, the object of the present invention, is partially shared with BC-2 and therefore recognizes an epitope located in the same protein region. This region is highly expressed in various tumor tissues. It is therefore important to obtain homogeneous monoclonal antibodies specific for this region that are used for cancer diagnosis or targeting.

  Furthermore, the present invention has shown that antibody ST2485 has the advantage of binding to tenascin in addition to antibody ST2146, and thus is useful in pretargeting methods involving the combination of two antibodies.

Summary of the Invention An anti-human tenascin monoclonal antibody has been found that solves the above problems. This is because the antibody does not have a non-functional light chain and provides an additive advantage when used in combination with another anti-tenascin antibody. This antibody is an object of the present invention along with the method of obtaining it and its use in therapy, particularly for use in the preparation of products useful for the treatment of diseases characterized by the expression of tenascin, eg tumors.

  The present invention relates to antibodies and antibody fragments that may contain additional markers and diagnostic agents, methods for obtaining the antibodies and antibody fragments, pharmaceutical compositions comprising the antibodies and fragments thereof, and diagnostic and therapeutic methods using them. As well as kits useful for carrying out the method.

  The invention also encodes the DNA encoding the antibody or fragment thereof, a vector comprising such a DNA, a host cell comprising such a vector, the nucleotide sequence, the protein encoded by SEQ ID NO: 1 and SEQ ID NO: 2, the protein and the fragment encoding DNA, specific complementarity determining regions (CDRs) and proteins containing such CDRs. In particular, the object of the present invention is also a hybridoma producing said monoclonal antibody.

  The antibodies that are the object of the present invention are characterized by the light and heavy chain variable region sequences of SEQ ID NO: 1 and SEQ ID NO: 2, shown in FIGS. 17 and 18, respectively. Briefly, the antibody that is the object of the present invention is identified by the name ST2485. Also, ST2485 fragments or chimeric or recombinant derivatives thereof can be produced and used within the scope of the present invention. The object of the present invention is also a proteolytic fragment of the antibody capable of binding to an antigenic epitope within the A1-4-D region of human tenascin. In the description of the present invention, an antibody fragment means a fragment capable of binding to an antigenic epitope within the A1-4-D region of human tenascin-C.

  According to the invention, the antibody or proteolytic fragment is preferably biotinylated.

  A further object of the present invention is a hybridoma cell line designated cST2485 that produces antibody ST2485.

  The hybridoma cell line was transferred to Centro di Biotecnologie Avanzate, Largo Rossana Benzi 10, Genoa, Italy on November 12, 2003 in Centro the Biotechnology Avanzate Largo Rossana Benzi 10. Genoa, Italy. Deposited as deposit number PD03003 by convention.

  The object of the present invention is also a recombinant derivative of antibody ST2485, which may be biotinylated. Specifically, preferred recombinant derivatives are those in which the mouse constant region is replaced by its human counterpart (Ferrer, C., et al., J. Biotechnol., 52: 51-60, 1996). Or a mouse constant region directed to a biologically active component, e.g. a member of the avidin family (Penichet ML., Manuel, L., et al., J. Immunol., 163: 44214426, 1999), tumor Substituted with a growth factor useful for stimulation of a given immunological effector (e.g., G-CSF, GM-CSF), or a mouse constant region is a pharmaceutically active ingredient, e.g., a superantigen, a toxin, Cytokines or any other protein useful for enhancing anti-cancer therapeutic efficacy (Di Massimo, AM, et al., British J. Cancer, 75 (6): 822-828, 1997; Parente D., et al., Anticancer Research, 17 (6A): 4073-4074, 1997). Methods for obtaining such derivatives are well known to those skilled in the art.

  A further object of the present invention is a conjugated derivative of antibody ST2485, which may be biotinylated.

  Specifically, preferred conjugated derivatives are those in which a biologically active component is bound to the antibody by conventional systems. Examples of biologically active compounds include members of the avidin family, growth factors useful for stimulating immunological effectors directed against tumors (e.g., G-CSF, GM-CSF), or mouse constant regions. , Pharmaceutically active ingredients such as super antigens, toxins, cytokines or any other protein useful for enhancing anti-cancer therapeutic efficacy, anti-cancer agents, and those substituted with radioisotopes.

  A further object of the present invention is preferably to a subject suffering from a cancer expressing tenascin, referred to as a three-step pretargeting, also known as the trademark PAGRIT®, of the antibody or derivative thereof. Use for the preparation of a pharmaceutical product useful in a method for cancer radioimmunotherapy preferably performed by a method comprising the administration of a biotinylated antibody ST2485 or a proteolytic fragment thereof.

  Recombinant derivatives of antibodies and conjugates thereof that are the object of the present invention can be advantageously used for cancer treatment. Therefore, the use of antibodies and fragments or derivatives thereof in the preparation of a medicament for the treatment of tumors expressing tenascin constitutes a further object of the present invention.

  For the implementation of radioimmunotherapy, two therapeutic kits are described, one for the whole body and the other for topical use. These kits are also known by the trademark PAGRIT®. The systemic kit consists of 5 vials containing: vial 1 containing a biotinylated antibody or fragment or derivative thereof according to the present invention; vial 2 containing avidin; vial 3 containing streptavidin; vial 4 containing biotinylated human albumin And vial 5 containing biotin DOTA (ie the biotin derivative described in WO 02/066075). The topical kit consists of three vials corresponding to vials 1, 2 and 5 of the whole body kit. Vials are prepared such that they are suitable for injection in a subject to be treated, preferably a human subject. For intraoperative treatment of patients undergoing surgical removal of the mass, using the reagents that are the object of the present invention alone or in combination with other components of the PAGRIT® kit for the implementation of radioimmunotherapy A method called IART (Intraoperative Avidination for Radionuclide Treament) is described.

  A particular container, preferably a container in the form of a vial suitable for injection containing the antibody or fragment thereof in biotinylated form constitutes a further object of the invention.

  According to one embodiment of the invention, in a therapeutic kit, the biotinylated antibody is combined with other anti-tenascin antibodies, preferably antibodies against the EGF-like region of the protein. Alternatively, the biotinylated antibody may be conjugated to other antibodies specific for the tumor. A general description of this type of method is described in EP 0 496 074, European Journal of Nuclear Medicine Vol. 26, No 4; April, 1999; 348-357, US 5,968,405.

  A further object of the present invention is the use of monoclonal antibody ST2485 for acquiring images for subsequent diagnostic purposes (“imaging”) by in vivo immunolocalization in tumors.

  A further object of the present invention is the use of monoclonal antibody ST2485 in combination with a second anti-tenascin antibody in a test for the production of a diagnostic kit for measuring the blood concentration of tenascin.

  These and other objects of the present invention are described in detail below with reference to examples and drawings.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1: Schematic representation of the strategy used to generate human tenascin-C and BC-2-like antibodies.

Figure 2: SDS-PAGE (a, b) and Western blot (c, d) analysis of antibody ST2485 under reducing and non-reducing conditions. Under reducing conditions, a double band is observed at the level of the antibody light chain (b, d).

Figure 3: SDS-PAGE analysis of antibody ST 2485 subjected to deglycosylation by digestion with the enzyme PNGaseF (Flavobacterium-derived peptide-N-glycosidase): disappearance of the high molecular weight light chain band is observed.

Figure 4: Hydroxyapatite chromatography profiles of antibodies ST2485 and BC-2.

Figure 5: Specific binding of ST2485 to human tenascin and Tn (A _(1-4) -D) fragments by Western blot analysis.

FIG. 6: Competition ELISA between ST2485 and BC-2 for binding to tenascin C (a) or Tn (A _(1-4) -D) fragment (b).

Figure 7: Immunoreactivity of ST2485 compared to BC-2 against tenascin C (a) and Tn (A _(1-4) -D) fragment (b).

Figure 8: Immunoreactivity of biotinylated ST2485 and BC-2 against tenascin C (a) and Tn (A _(1-4) -D) fragment (b).

Figure 9: Cross-reactivity of antibody ST2485 with mouse tenascin expressed in LMM3 tumor (mouse breast cancer) and normal mouse intestine.

Figure 10: Protocol adopted for ST2485 biodistribution studies in nude mice.

Figures 11 and 11a: Biodistribution of ST2485 at various doses compared to BC-2. At all doses administered, ST2485 accumulation in the tumor is at least twice that of BC-2.

Figures 12 and 12a: Tumor to non-tumor ratio of ST2485 compared to BC-2. These ratios are higher for antibody ST2485 at all doses administered.

FIG. 13: In vitro interference (a) and additive test (b) of anti-tenascin antibodies ST2485 and ST2146 by ELISA test.

Figures 14 and 14a: In vitro additivity test of antibodies ST2485 and ST2146 by BiaCore analysis.

FIG. 15: Scheme adopted for in vivo additive studies of antibodies ST2485 and ST2146.

FIG. 16: In vivo additivity of antibodies ST2485 and ST2146, expressed in ng of antibody localized per gram of tumor.

Figure 17: SEQ ID NO: 1, ST2485 light chain variable region (VL) sequence. The encoded amino acid sequence is shown in SEQ ID NO: 3.

FIG. 18: SEQ ID NO: 2, ST2485 heavy chain variable region (VH) sequence. The encoded amino acid sequence is shown in SEQ ID NO: 4.

Detailed Description of the Invention We have prepared a novel anti-human tenascin antibody designated ST2485 produced from the hybridoma cell line cST2485, whose light and heavy chain variable region sequences are SEQ ID NO: 1 and SEQ ID NO: 1, respectively. 2. The antibody can bind to an antigenic epitope within the A _(1-4) -D region of human tenascin.

Proteolytic fragments of antibodies according to the present invention are capable of binding to an antigenic epitope within the A _(1-4) -D region of human tenascin.

  The recombinant derivatives of the antibodies according to the invention are obtained according to conventional methods well known to those skilled in the art. Preferred recombinant derivatives are those in which the mouse constant region is replaced by its human counterpart, or a biologically or pharmaceutically active component, or a member of the avidin family.

  According to the present invention, conjugated derivatives are obtained by conventional methods well known in the art. Preferred conjugated derivatives are those in which a biologically active moiety is attached to the antibody. Examples of biologically active moieties include members of the avidin family, growth factors useful for stimulating immunological effectors directed at tumors (eg, G-CSF, GM-CSF), pharmacologically active Moieties such as toxins, superantigens, cytokines or any other protein useful for enhancing therapeutic anticancer effects, anticancer agents, and radioisotopes.

  See WO 03/072608 for further information regarding the preparation of recombinant and conjugated derivatives.

  According to the present invention, recombinant or conjugated derivatives of monoclonal antibodies are also referred to as “derivatives”.

  In one preferred embodiment of the invention, the antibodies, fragments and derivatives thereof may be biotinylated.

  The antibodies, fragments and derivatives thereof according to the invention may also advantageously contain further markers and diagnostic agents.

  The present invention also includes DNA encoding the above monoclonal antibody or a fragment thereof. The present invention also includes a vector containing the DNA and a host cell containing the vector.

  Vectors and host cells are within the well-known facts belonging to this field.

  The present invention also includes the nucleotide sequence, the protein encoded by SEQ ID NO: 1 and SEQ ID NO: 2 or fragments thereof, and the DNA encoding it.

  The present invention also includes a specific CDR (complementarity determining region) of the antibody and a protein containing the CDR.

The method for preparing an antibody according to the invention comprises the following steps:
a) immunization of an animal, preferably a mouse, with an A _(1-4) -D fragment of human tenascin;
b) fusion of the animal's somatic spleen cells with myeloma cells that do not produce immunoglobulins;
c) Selection of monoclonal antibodies.

  Monoclonal antibody ST2485 is produced by the hybridoma cell line cST2485 described above.

  According to the present invention, an antibody or fragment or derivative thereof, which may be biotinylated, is used for the preparation of a pharmaceutical product useful for the treatment and diagnosis of diseases characterized by the expression of tenascin. Specifically, the disease is a tumor, and more specifically includes glioma, breast cancer, lung cancer, fibrosarcoma and squamous cell carcinoma.

  In one preferred aspect, the pharmaceutical product is used in a two-stage perioperative therapy for solid tumors as described in International Patent Application WO 03/07268. In this type of therapy, a biotinylated antibody and then avidin are administered, constructing an “artificial receptor” for the actual next administered anticancer agent. In this case, the anticancer agent is delivered by biotin, which is contained in a compound, hereinafter referred to as a biotin compound, suitable for forming a complex with the anticancer agent and administered systemically in the postoperative period. Biotin is actually localized only where avidin is present, in which case it is introduced by the surgeon a few hours (e.g., 4 to 72 hours) early during surgery, which is of interest for treatment. You can be sure that avidin exists in a certain area. This is advantageous in that it dramatically reduces the elapsed time between removal of the primary tumor and adjuvant therapy.

  With respect to the industrial aspect of the present invention, the antibodies described herein are suitably formulated into pharmaceutical compositions or therapeutic or diagnostic kits according to common practice in the pharmaceutical field.

  The pharmaceutical compositions and kits are of the conventional type in the art and can be prepared by one skilled in the art only based on well-known facts. Examples of pharmaceutical compositions are provided in the literature cited in the present invention. The same applies to the kit. Particularly preferred are tumor radioimmunotherapy kits as described by Paganelli et al and in the above studies in EP 0 496 074, WO 02/066075, WO 03/072608 and WO 03/075960. Particular use of the radioimmunotherapy kit can be carried out by the device described in WO 03/069632.

  Also included within the scope of the invention is a pharmaceutical composition comprising an antibody or derivative or fragment thereof, which may be biotinylated, in a mixture with at least one pharmaceutically acceptable excipient or vehicle.

  In a particular embodiment of the invention, the kit is provided for systemic radioimmunotherapy, in particular three-stage pretargeted radioimmunotherapy, comprising 5 vials, wherein vial 1 may be an biotinylated antibody or its Including fragment or recombinant derivative or conjugate or analog thereof; vial 2 contains avidin; vial 3 contains streptavidin; vial 4 contains biotinylated human albumin; and vial 5 contains biotin DOTA.

  In another embodiment of the invention, the kit is for local area radioimmunotherapy and comprises three vials that are the same as vials 1, 2 and 5 of the systemic kit.

(I)
［式中、
Qは、-(CH₂)n-基、ここで、nは4〜12の整数であり、この場合、R'は存在しない、または、
Qは、-(CH₂)_a-CH(R')_b-(CH₂)_b-からなる群から選択され、ここで、aおよびbは独立に0〜nの整数であり、R'は以下に定義する通りである、または、
Qは、シクロヘキシル、フェニルであり、この場合、R'はシクロヘキシルまたはフェニル環上の置換基である;
Rは、水素または-Λ、ここで-Λは式(II)の大環状分子、

(II)
ここで、様々なYは、同一であっても異なっていてもよく、水素、直鎖状または分枝状C₁-C₄アルキル、-(CH₂)_m-COOHからなる群から選択され、ここでmは1〜3の整数である、
Xは、水素、または基-CH₂-U、ここでUは、メチル、エチル、およびp-アミノフェニルからなる群から選択され、または、
Xは、基-(CHW)_o-Zであり、ここでoは1〜5の整数であり、Wは、水素、メチルまたはエチル、Zは、O、N-R₁およびSから選択される１以上のヘテロ原子を含む5-または6-員複素環基であり、ここで、R₁は水素または直鎖状または分枝状C₁-C₄ アルキルである;またはZは、-NH₂、-NH-C(=NH)-NH₂、または-S-R₂からなる群から選択され、ここでR₂は直鎖状または分枝状C₁-C₄ アルキルである;
pは2または3の整数である;
R'は、水素、直鎖状または分枝状C₁-C₄ アルキル、-(CH₂)_q-Tからなる群から選択され、ここで、Tは、-S-CH₃、-OH、-COOHからなる群から選択され、そして、qは1または2の整数である;
R''はR'と同義であるが、その上に以下の条件に従う: Rが-Λの場合、R''は水素である; Rが水素の場合、R''は-Λである、あるいはRおよびR''は、それぞれ、(Rについて)-(CH₂)_r-Λおよび(R'について)-Λであり、ここでrは4〜12の整数である、ここで、Qは、-(CH₂)_n- 基であり、ここでnは4〜12の整数である］。 In a particularly preferred embodiment, the biotin DOTA in a vial containing biotin DOTA is a compound of formula (I).

(I)
[Where:
Q is, - (CH ₂₎ n- group, where, n is 4 to 12 integer, in this case, R 'is absent, or,
Q is selected from the group consisting of — (CH ₂ ) _a —CH (R ′) _b — (CH ₂ ) _b —, wherein a and b are independently integers from 0 to n, and R ′ is As defined below, or
Q is cyclohexyl, phenyl, where R ′ is a substituent on the cyclohexyl or phenyl ring;
R is hydrogen or -Λ, where -Λ is a macrocycle of formula (II),

(II)
Where the various Ys, which may be the same or different, are selected from the group consisting of hydrogen, linear or branched C ₁ -C ₄ alkyl, — (CH ₂ ) _m —COOH, Where m is an integer from 1 to 3,
X is hydrogen, or a group —CH ₂ —U, where U is selected from the group consisting of methyl, ethyl, and p-aminophenyl, or
X is a group — (CHW) _o —Z, where o is an integer from 1 to 5, W is hydrogen, methyl or ethyl, Z is one or more selected from O, NR ₁ and S 5- or 6-membered heterocyclic groups containing heteroatoms, wherein R ₁ is hydrogen or linear or branched C ₁ -C ₄ alkyl; or Z is —NH ₂ , — NH-C (= NH) -NH 2, or it is selected from the group consisting of -SR _2, wherein R ₂ is a linear or branched C ₁ -C ₄ alkyl;
p is an integer of 2 or 3;
R ′ is selected from the group consisting of hydrogen, linear or branched C ₁ -C ₄ alkyl, — (CH ₂ ) _q —T, wherein T is —S—CH ₃ , —OH, Selected from the group consisting of -COOH and q is an integer of 1 or 2;
R '' is synonymous with R 'but subject to the following conditions: R''is hydrogen when R is -Λ; R''is -Λ when R is hydrogen; Or R and R '' are (for R)-(CH ₂ ) _r -Λ and (for R ′)-Λ, respectively, where r is an integer from 4 to 12, where Q is , - (CH _{2) n} - group, wherein n is 4 to 12 integer.

  These compounds are described in WO 02/066075.

In a particularly preferred embodiment, in a vial containing avidin, the avidin is an avidin dimer in which two molecules of avidin are linked via a —NH ₂ group by suberate, or two molecules of avidin are described in WO 03/075960 In this way, it is an avidin dimer that is bonded via a -COOH group with polyethylene glycol having a molecular weight of 3,400.

  In the kit according to the invention, the optionally biotinylated antibody, its proteolytic fragment, its derivative may preferably be present in combination with other anti-tenascin antibodies that target the EGF-like region of the protein. And may be present in combination with other tumor-specific antibodies.

  All aspects of the present invention apply to tumor diagnosis, particularly tumor immunolocalization methods such as imaging, in addition to application to therapy of tumors expressing tenascin.

  A further object of the invention is a container containing an antibody or proteolytic fragment thereof or derivative thereof, preferably in the form of a vial suitable for injection, which may be biotinylated and / or radiolabeled.

In another aspect of the invention, the antibody or fragment, recombinant derivative, or analog is used for the purpose of measuring circulating tenascin levels, particularly isoforms comprising the A _(1-4) -D region. Used in combination with a second tenascin-specific antibody in a sandwich in vitro ELISA assay under conditions where the antibody binds to a second antigenic epitope of tenascin.

The following examples further illustrate the present invention.
Example 1
For the purpose of creating new hybridomas with BC-2 specificity but not producing non-functional light chains, Balb / c mice were isolated from human tenascin A _(1-4) -containing the epitope recognized by BC-2. Immunized with the D fragment (Siri, A., et al., Nucl. Acid Res., 19 (3): 525-531, 1991; Balza, E., et al., FEBS, 332: 39-43, 1993 ). A schematic of the strategy used to generate human tenascin-C and BC-2-like antibodies is shown in FIG. According to standard methods, spleen cells of mice immunized with Tn (A _(1-4) -D) fragments are fused with non-immunoglobulin producing myeloma cells Sp2 / 0-Ag14 (Cianfriglia, M., et al , Methods Enzymol., 121: 193-210, 1986), the resulting hybridoma population was subjected to screening by ELISA test for purified tenascin from SK-Mel 28 human melanoma cells (ICLC HTL99010). Hybridomas secreting anti-tenascin antibodies were cloned twice by limiting dilution in growth medium containing FCS and twice in animal-free protein-free medium (Animal Derived Component Free Medium HyClone, HyQR Perbio).

  Production of ST2485 standards was achieved by culturing cST2485 hybridoma cells in a 2-liter bioreactor; by sub-clone cST2485 / A3e / by double serial limiting dilution subcloning of cST2485 Post Production Cell Bank (PPCB) A12f was selected and used to generate Master Cell Bank (MCB) and Working Cell Bank (WCB).

  ST2485 is a mouse immunoglobulin of the IgG1 / k isotype.

  FIG. 2 shows SDS-PAGE (a, b) and Western blot (c, d) analysis of antibody ST2485 under reducing and non-reducing conditions. Under reducing conditions (b, d), a double band is observed at the level of the antibody light chain; Western blot analysis indicates that the two bands are immunoreactive as immunoglobulin light chains. Hydroxyapatite chromatography analysis of antibody ST2485, shown in FIG. 3, confirms that the antibody is slightly heterogeneous with a slightly asymmetric peak. On the other hand, in the same analysis, antibody BC-2 shows a significantly heterogeneous profile, where three peaks are distinguishable, of which only peak 1 corresponds to a fully functional homogeneous antibody. .

  In order to define the heterogeneity of antibody ST 2485, a sample of antibody ST 2485 was subjected to digestion of N-glycoside residues with the enzyme PNGaseF (flavobacterium-derived peptide-N-glycosidase). Enzymatic digestion was performed using the Prozyme deglycosylation kit (Cat.No.GE51001) under the conditions indicated by the manufacturer: Approximately 8 μg of ST2485 and 5 mU of enzyme were reacted overnight at 37 ° C. in a 10 μl reaction mixture. It was. Digested or undigested antibody ST2485 was run on a 12% polyacrylamide gel under reducing conditions. The gel was stained with Coomassie brilliant blue.

  As shown by SDS-PAGE analysis under reducing conditions shown in FIG. 4, the high molecular weight light chain band disappeared upon digestion. This finding indicates that the two light chain bands of antibody ST2485 correspond to its glycosylated (high molecular weight) and non-glycosylated (low molecular weight) variants. The presence of potential N-glycosylation sites was also confirmed by immunoglobulin cDNA sequence data (Figure 17).

  As described in M. Sassano et al. Nucl. Ac. Res. (1994) 22, 1768-1769, the variable region of the kappa light chain was extracted from the circular cDNA using the following primer pairs that recognize the constant region of the antibody Amplified (5 ′ GGGAAGATGGATACA-GTTGGTG (SEQ ID NO: 5), 5 ′ CAAGAGCTTCAACAGGAATGAG (SEQ ID NO: 6)).

As shown by the Western blot analysis shown in FIG. 5, the binding of ST2485 to the tenascin-A _(1-4) -D region is specific. The antibody bound to human tenascin and the A _(1-4) -D fragment, but not to the EGF-like fragment of the same protein containing the epitope recognized by the BC-4 antibody.

ST2485 was shared by human tenascin, in a unique epitope, or partially with BC-2, as shown by a competitive ELISA assay between the two antibodies shown in FIG. 6, antibody ST2485 and BC-2 antibody Bind at the epitope. Antigen tenascin C (a) or Tn A ₍ with biotinylated antibody BC-2 at concentrations established in preliminary experiments, with increasing concentrations of non-biotinylated BC-2 (curve 1) or ST2485 (curve 2) _{1-4) The} solution was dispensed on the plate sensitized with the -D fragment (b). Biotinylated antibody binding is measured after addition of HRP-streptavidin and the associated chromogenic substrate TMB. Antibody ST2485 resulted in 40% inhibition of binding of BC-2 to tenascin-C or TnA _(1-4) -D fragment.

As shown in FIG. 7, ST2485 immunoreactivity was assessed in comparison to BC-2 by ELISA assay against tenascin C and Tn A _(1-4) -D fragments. Antibodies ST2485 and BC-2 and normal mouse immunoglobulin (nMIgG) were dispensed at increasing concentrations on plates sensitized with tenascin C (a) or Tn A _(1-4) -D fragment (b); The dose response curve of the antibody could be determined by the addition of secondary anti-mouse antibody labeled with alkaline phosphatase and related chromogenic substrate (pNPP). The amount of ST2485 required to obtain OD 1.0 is approximately one-third of the amount of BC-2 for total tenascin (a) and BC-2 for Tn (A1-4-D) fragment (b) About one-tenth of the amount.

The affinity of antibody ST2485 for both tenascin C and Tn (A _1-4 -D) fragments was assessed by BIAcore analysis. The KD ₁ of ST2485 for Tenascin C is 9.77 x 10 ^-10 M (ka ₁ = 6.02 x 10 ⁵ ; kd ₁ = 5.88 x 10 ^-4 ), and the KD _{1 of} BC-2 is 2.54 x 10 ^-7 M (ka ₁ = 9.85 x 10 ³ ; kd ₁ = 2.5 x 10 ^-3 ). The KD ₁ of ST2485 for the Tn (A _1-4 -D) fragment is 9.72 x 10 ^-10 M (ka ¹ = 3.28 x 10 ⁵ ; kd1 = 3.19 x 10 ^-4 ), and the KD _{1 of} BC-2 is 7.39. x 10 ^-8 M (ka ₁ = 2.68 x 10 ⁴ ; kd ₁ = 1.93 x 10 ^-3 ).

Since maintaining immunoreactivity after biotinylation is a basic requirement for antibodies used in pretargeting, the behavior of biotinylated ST2485 (8.3 biotin / molecule) can be compared to biotinylated BC-2 (7.6 biotin / molecule). As compared to molecules) by ELISA and BIAcore assays. Referring to Figure 8, biotinylated and non-biotinylated antibodies ST2485 and BC-2 and normal mouse immunoglobulin (nMIgG) were sensitized with increasing concentrations of tenascin C (a) or Tn A _(1-4) -D fragment (b). A dose response curve of each biotinylated antibody compared to non-biotinylated antibody by adding secondary anti-mouse antibody labeled with alkaline phosphatase and related chromogenic substrate (pNPP). Judgment (residual immunoreactivity) became possible. The residual immunoreactivity of biotinylated ST2485 and BC-2 to tenascin-C was 76% and 88%, respectively. The remaining immunoreactivity of ST2485 and BC-2 against the Tn A _(1-4) -D fragment was 73% and 83%, respectively.

BIAcore analysis of biotinylated antibody against whole tenascin shows that for biotinylated ST2485, KD ₁ affinity = 2.88 x 10 ^-9 M (ka ₁ = 2.8 x 10 ⁵ ; kd ₁ = 8.07 x 10 ^-4 ) and biotinylated BC-2 KD ₁ affinity = 3.71 x 10 ^-7 M (ka ₁ = 6.0 x 10 ³ ; kd ₁ = 2.23 x 10 ^-3 ). Thus, biotinylated ST2485 maintained good immunoreactivity and affinity characteristics and retained approximately 100 times the affinity for tenascin compared to biotinylated BC-2.

  Immunohistochemical studies on various human tumors (breast, lung, stomach, colon) showed that ST2485 localization was at the level of the extracellular matrix, similar to the report for BC-2 (Natali PG, et al., Int. J. Cancer, 47: 811-16, 1991). These studies also showed the ability of ST2485 to have cross-reactivity with mouse tenascin, including those expressed in normal mouse tissues, as seen in FIG. LMM3 tumors fixed in formalin (mouse breast cancer) and normal mouse intestine sections were incubated with 10 μg / ml ST2485 or normal mouse IgG1 antibody (control). After incubation with a biotinylated secondary anti-mouse antibody and avidin-biotin-peroxidase complex (Vectastain Elite ABC kit), binding to tenascin was detected by addition of the chromogenic substrate DAB (Vector). Controls were performed using Mayer's hematoxylin. LMM3 and mouse intestinal sections are positive for ST2485 but not for the control antibody. These studies compared to BC-2 could not be performed because antibody BC-2 cannot recognize paraffin-embedded fixed tissue sections. The ability of ST2485 to recognize mouse tenascin, including those expressed by normal tissues, makes the results of interest in antibody biodistribution studies in mouse models described below important.

In order to evaluate the ability of the antibody to localize to the mass, biodistribution studies of biotinylated ¹²⁵ I-labeled ST2485 and BC-2 were performed. These studies were conducted in nude mice transplanted with human tumors expressing tenascin according to the protocol schematically shown in FIG.

Animals were inoculated subcutaneously with 4 × 10 ⁶ human colon cancer HT-29 cells in 0.1 ml sterile saline. After 15 days, when the tumor size reaches approximately 200 mg, groups of 5 animals are administered ¹²⁵ I-BC2, ¹²⁵ I-ST2485, or normal mouse ¹²⁵ I-immunoglobulin (nMIg) intravenously, All antibodies were biotinylated (7-9 biotin / mol) and were administered at 5 doses: 0.2-0.5-1-2-5 μg / mouse in 100 μl sterile PBS. Five days after antibody administration, animals were sacrificed and blood, spleen, kidney, liver and tumor samples were taken and examined for the presence of radioactivity. Twenty-four hours prior to sacrifice, animals were administered intravenously with 100 μl of avidin in sterile PBS at a 100-fold dose of antibody (follow-up).

  The results shown in FIGS. 11 and 11a indicate that both BC-2 and ST2485 are specifically localized to the tumor regardless of dose (the amount of antibody is expressed as a percentage of the dose injected per gram of tissue: % ID / gr). Furthermore, ST2485 was more localized to the tumor compared to BC-2 at all doses assayed and always maintained a higher tumor to non-tumor ratio (FIGS. 12 and 12a). In particular, ST2485 accumulation in tumors at a dose of 1 μg / mouse was 16% of the injected dose, while BC-2 accumulation did not exceed 5% at any of the doses assayed. At the 1 μg / mouse dose, the tumor: blood ratio for ST2485 exceeded 20 (optimum dose), whereas for antibody BC-2 this ratio did not exceed 6 at any of the doses assayed. In other organs, the tumor to non-tumor ratio of ST2485 at a dose of 1 μg / mouse was 9, 26, and 43 for spleen, kidney, and liver, respectively. On the other hand, for BC-2, these ratios were always below 7 at any dose assayed. Tumor to non-tumor ratios are shown in FIGS. 12 and 12a.

  Since ST2416 was created against an epitope localized in the EGF-like region of the protein (De Santis, R., et al., Br. J. Cancer, 88: 996-1003, 2003, WO 03 / 072608), in vitro and in vivo additive experiments were performed on these two antibodies in order to evaluate the possibility of combining anti-tenascin antibodies ST2485 and ST2146 in PAGRIT® and IART methods. Figure 13 shows the absence of epitope interference between antibodies ST2485 and ST2146 (13a) and the additive binding to their antigens in two ELISA tests performed on plates sensitized with tenascin-C. (13b). To assess interference, (a) different concentrations of biotinylated antibody ST2485 were distributed on microplates sensitized with tenascin C in the absence (1) and presence (2) of saturating concentrations of ST2146 : Overlapped curves 1 and 2 show the absence of epitope interference between the two antibodies. This is because the binding of biotinylated ST2485 to tenascin is not affected by the presence of ST2146 (and vice versa; data not shown). Curve 3 shows the signal obtained when biotinylated ST2485 was seeded in the presence of saturated non-biotinylated ST2485 (control).

  FIG. 13b shows an additive test in which biotinylated antibodies ST2485 and ST2146 were dispensed at saturated doses alone or together in microplates sensitized with tenascin-C: the mixture of the two antibodies is the sum of the signals Yielded the same signal and thus the binding of these two antibodies was shown to be additive.

  The in vitro additivity of these two antibodies was also assayed using BIACore analysis as shown in FIGS. 14 and 14a. Tenascin was immobilized on a chip (CM5 sensor chip, Biosense) according to the method described (De Santis R .; et al .; Br. J. Cancer, 88: 996-1003, 2003). Two consecutive injections of the first antibody were administered at a saturating concentration (10 μM) (ST2485 in a, ST2146 in c) followed by a second anti-tenascin antibody (ST2146 in a, ST2485 in c). Sensorgrams, a) and c) show that these two antibodies can bind to each of the corresponding epitopes additively, as indicated by the increase in resonance signal produced by each single antibody. Show. Sensorgrams b) and d) show injections of ST2485 and ST2146, which were later administered with isotypical control antibodies, mIgG2b and mIgG1, respectively.

  Additivity studies of the two antibodies ST2485 and ST2146 were also performed in vivo in previously described animal models. The study scheme is shown in FIG. 15 and the results obtained, expressed as ng antibody per gram of tumor, are shown in FIG. The data confirms that the two antibodies are also additive in the animal model. The mixture of two labeled antibodies actually showed accumulation in the tumor reaching 93% of the theoretical value (the mathematical sum of the two individual labeled antibodies). A group of control animals was treated with a mixture of radiolabeled antibody and a second “non-radioactive” antibody to avoid interference.

  Primer pair (5 'GGGAAGATGG-ATACAGTTGGTG 3' (SEQ ID NO: 5), 5 'CAAGAGCTTCAACAGGAAT-GAG 3' (SEQ ID NO: 6) starting from the circular cDNA of the variable region of the kappa light chain ) Was used as described in M. Sassano, et al., Nucl. Ac. Res., (1994) 22, 1768-1769.

  Primer pairs (5 'ATGGAGTTAG-TTTGGGCAGCAG 3' (SEQ ID NO: 7), 5 'GCACAACCACCATACTGAG-AAG 3' (SEQ ID NO: 8) ) Was used as described in M. Sassano, et al., Nucl. Ac. Res., (1994) 22, 1768-1769.

  FIG. 17 shows SEQ ID NO: 1, which is the sequence of the light chain variable region (VL) of ST2485.

  FIG. 18 shows SEQ ID NO: 2, which is the heavy chain variable region (VH) sequence of ST2485.

Comparative characterization of ST2485 compared to BC-2 showed that antibody ST2485 has the following characteristics:
Antibody ST2485 recognizes an epitope within the A _(1-4) -D region of human tenascin close to or partially overlapping the epitope of BC-2;
-Antibody ST2485 is an antibody that is homogeneous with respect to the composition of the light and heavy chains in that the observed heterogeneity is due to light chain glycosylation variants;
-Antibody ST2485 is more immunoreactive than BC-2;
Antibody ST2485 has a higher affinity for antigen than BC-2;
-Antibody ST2485 is comparable to BC-2 in maintaining immunoreactivity after biotinylation;
-Antibody ST2485 is superior to BC-2 for tumor localization in animal models;
Antibody ST2485 binds to tenascin C additively with anti-tenascin monoclonal antibody ST2146 both in vitro and in vivo in animal models.

References
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Schematic representation of the strategy used to generate human tenascin-C and BC-2-like antibodies. SDS-PAGE (a, b) and Western blot (c, d) analysis of antibody ST2485 under reducing and non-reducing conditions. Under reducing conditions, a double band is observed at the level of the antibody light chain (b, d). SDS-PAGE analysis of antibody ST 2485 subjected to deglycosylation by digestion with the enzyme PNGaseF (Flavobacterium-derived peptide-N-glycosidase): disappearance of the high molecular weight light chain band is observed. Hydroxyapatite chromatography profiles of antibodies ST2485 and BC-2. Binding specificity of ST2485 for human tenascin and Tn (A _(1-4) -D) fragments by Western blot analysis. Competition ELISA between ST2485 and BC-2 for binding to tenascin C (a) or Tn (A _(1-4) -D) fragment (b). ST2485 immunoreactivity compared to BC-2 against tenascin C (a) and Tn (A _(1-4) -D) fragment (b). Immunoreactivity of biotinylated ST2485 and BC-2 against tenascin C (a) and Tn (A _(1-4) -D) fragments (b). Cross-reactivity of antibody ST2485 with mouse tenascin expressed in LMM3 tumor (mouse breast cancer) and normal mouse intestine. Protocol adopted for ST2485 biodistribution study in nude mice. Biodistribution of ST2485 at various doses compared to BC-2. Biodistribution of ST2485 at various doses compared to BC-2. ST2485 tumor-to-non-tumor ratio compared to BC-2. ST2485 tumor-to-non-tumor ratio compared to BC-2. In vitro interference (a) and additive test (b) of anti-tenascin antibodies ST2485 and ST2146 by ELISA test. In vitro additive test of antibodies ST2485 and ST2146 by BiaCore analysis. In vitro additive test of antibodies ST2485 and ST2146 by BiaCore analysis. Scheme adopted for in vivo additive studies of antibodies ST2485 and ST2146. In vivo additivity of antibodies ST2485 and ST2146 expressed in ng of antibody localized per gram of tumor. SEQ ID NO: 1, ST2485 light chain variable region (VL) sequence. The encoded amino acid sequence is shown in SEQ ID NO: 3. SEQ ID NO: 2, ST2485 heavy chain variable region (VH) sequence. The encoded amino acid sequence is shown in SEQ ID NO: 4.

Claims (32)

An anti-human tenascin monoclonal antibody whose light and heavy chain variable region sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively;
Light and heavy chain variable, which can bind to an antigenic epitope within the A _(1-4) -D region of human tenascin, including the variable region of antibodies to the A _(1-4) -D region of human tenascin A proteolytic fragment of the anti-human tenascin monoclonal antibody whose region sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively;
Light and heavy chain variable, which can bind to an antigenic epitope within the A _(1-4) -D region of human tenascin, including the variable region of antibodies to the A _(1-4) -D region of human tenascin A recombinant derivative of the anti-human tenascin monoclonal antibody, wherein the region sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively, wherein the recombinant derivative is a mouse constant region substituted with its human counterpart or avidin Substituted with a biologically or pharmaceutically active ingredient selected from the group consisting of family members, growth factors, superantigens, toxins and cytokines); or within the A _(1-4) -D region of human tenascin A group of avidin family members, growth factors, toxins, superantigens, cytokines, anticancer agents and radioisotopes The selected biologically active component is a light and heavy chain variable region conjugated to the anti-human tenascin monoclonal antibody whose light and heavy chain variable region sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. A conjugated derivative of the anti-human tenascin monoclonal antibody whose sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively.   The antibody, proteolytic fragment, recombinant derivative, or conjugated derivative of claim 1, which is derived from a mouse.   3. The antibody fragment of claim 1 or 2 conjugated with an additional tumor marker and tumor diagnostic agent.   The recombinant derivative of the antibody of claim 2, wherein the mouse constant region is replaced by its human counterpart.   The recombinant derivative of an antibody of claim 2, wherein the mouse constant region is replaced by a biologically active component selected from the group consisting of avidin family members, growth factors, superantigens, toxins and cytokines. Mouse constant regions are substituted by the pharmaceutically active ingredient selected from the superantigen, a toxin, and cytokine or Ranaru group, recombinant derivatives of the antibody of claim 2.   The recombinant derivative of the antibody of claim 2, wherein the mouse constant region is replaced by a member of the avidin family.   3. The antibody derivative of claim 1 or 2 conjugated to a biologically active substance selected from the group consisting of avidin family members, growth factors, toxins, superantigens, cytokines, anticancer agents and radioisotopes.   The antibody or proteolytic fragment or recombinant or conjugated derivative of claim 1 or 2 or the fragment of claim 3 or the derivative of any of claims 4-8 that is biotinylated and / or radiolabeled.   A DNA encoding the antibody of claim 1 or 2 or the fragment of claim 3.   A vector comprising the DNA of claim 10.   A host cell comprising the vector of claim 11.   The light and heavy chain variable region sequences each comprise an anti-human tenascin monoclonal antibody encoded by the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2, or the variable region of an antibody, respectively, wherein the light chain and heavy chain variable region sequences are Fragments of anti-human tenascin monoclonal antibodies that are SEQ ID NO: 1 and SEQ ID NO: 2.   DNA encoding the protein of claim 13 or a fragment thereof.   Producing the antibody of claim 1 or 2 deposited at Centro the Biotechnology Avanzate, Largo Rossana Benz 10, Genoa, Italy on 12 November 2003 under the accession number PD03003 in accordance with the Budapest Treaty Hybridoma.   Kit for systemic radioimmunotherapy comprising the following five vials: antibody or proteolytic fragment or recombinant or conjugated derivative of claim 1 or 2, or fragment of claim 3 or claim 4-8 A vial 1 containing the antibody or proteolytic fragment or recombinant or conjugated derivative of claim 9; a vial 2 containing avidin; a vial 3 containing streptavidin; a vial 4 containing biotinylated human albumin; And vial 5 containing biotin DOTA. 17. The kit of claim 16 for three-stage pretargeted radioimmunotherapy.   Kit for local area radioimmunotherapy consisting of the following three vials; antibody or proteolytic fragment or recombinant or conjugated derivative of claim 1 or 2, or fragment of claim 3 or claim 4-8 A vial 1 comprising any of the derivatives or the antibody or proteolytic fragment of claim 9 or a recombinant or conjugated derivative; vial 2 comprising avidin; and vial 3 comprising biotin DOTA. A kit according to any of claims 16 to 18 , wherein said biotin DOTA in vial 5 or vial 3, respectively, is a compound of formula (I):

(I)
[Where:
Q is, - (CH ₂₎ a n- group, where n is 4 to 12 integer, in this case R 'does not exist, or,
Q is selected from the group consisting of-(CH ₂ ) _a -CH (R ') _b- (CH ₂ ) _b- , where a and b are independently integers from 0 to n, and R' is Or as defined in
Q is cyclohexyl, phenyl, where R ′ is a substituent on the cyclohexyl or phenyl ring;
R is hydrogen or -Λ, where -Λ is a macrocycle of formula (II),

(II)
Where the various Ys may be the same or different and are selected from the group consisting of hydrogen, linear or branched C ₁ -C ₄ alkyl, — (CH ₂ ) _m —COOH, Where m is an integer from 1 to 3,
X is hydrogen or a group -CH ₂ -U,, wherein, U is selected from the group consisting of methyl, ethyl, and p- aminophenyl, or,
X is a group-(CHW) _o -Z, where o is an integer from 1 to 5, W is hydrogen, methyl or ethyl, and Z is selected from O, NR ₁ and S Or a 5- or 6-membered heterocyclic group containing one or more heteroatoms, wherein R ₁ is hydrogen or a linear or branched C ₁ -C ₄ alkyl; or Z is Selected from the group consisting of —NH ₂ , —NH—C (═NH) —NH ₂ , or —SR ₂ , wherein R ₂ is a linear or branched C ₁ -C ₄ alkyl;
p is an integer of 2 or 3;
R ′ is selected from the group consisting of hydrogen, linear or branched C ₁ -C ₄ alkyl, — (CH ₂ ) _q —T, wherein T is —S—CH ₃ , —OH, Selected from the group consisting of -COOH and q is an integer of 1 or 2;
R '' is synonymous with R 'but subject to the following conditions: R''is hydrogen when R is -Λ; R''is -Λ when R is hydrogen Or R and R '' are-(CH ₂ ) _r -Λ (for R) and -Λ (for R '), respectively, where r is an integer from 4 to 12, where Q is a — (CH ₂ ) _n — group, where n is an integer from 4 to 12. The vial 2 comprises avidin dimer in which the two avidin molecules are bound via the -NH ₂ group by suberate, 16. - any kit 19. The vial 2 comprises avidin dimer in which the two avidin molecules are bound via the -COOH groups by polyethylene glycol of molecular weight 3,400, according to claim 16 - one of the kit 19. 10. The antibody or proteolytic fragment or recombinant or conjugated derivative of claim 1 or 2, or the fragment of claim 3, or any derivative of claim 4-8 or the antibody or proteolytic fragment or set of claim 9. recombinant derivatives or joining derivatives is combined with another anti-tenascin antibody, claim 16 - 18 either kit. 23. The kit of claim 22 , wherein another anti-tenascin antibody is an antibody that targets the EGF-like region of the protein. 10. The antibody or proteolytic fragment or recombinant or conjugated derivative of claim 1 or 2, or the fragment of claim 3, or any derivative of claim 4-8 or the antibody or proteolytic fragment or set of claim 9. recombinant derivatives or joining derivatives is combined with another tumor specific antibody, according to claim 16 - 18 either kit.   10. The antibody or proteolytic fragment or recombinant or conjugated derivative of claim 1 or 2, or the fragment of claim 3, or any derivative of claim 4-8 or the antibody or proteolytic fragment or set of claim 9. A composition for a tumor immunolocalization method comprising a modified derivative or a conjugated derivative.   10. The antibody or proteolytic fragment or recombinant or conjugated derivative of claim 1 or 2, or the fragment of claim 3, or any derivative of claim 4-8 or the antibody or proteolytic fragment or set of claim 9. A container comprising a replacement derivative or a conjugated derivative. 27. The container of claim 26 , which is in the form of a vial.   3. The antibody or proteolytic fragment or recombinant or conjugated derivative of claim 1 or 2, or the fragment of claim 3 for administration to a human suffering from, or predicted to suffer from a tumor, or A composition used in a tumor imaging method for tumor detection, comprising the derivative of any one of claims 4 to 8, or the antibody or proteolytic fragment of claim 9 or a recombinant or conjugated derivative. 29. The composition of claim 28 , wherein the antibody or proteolytic fragment or recombinant or conjugated derivative is radiolabeled.   Combining the antibody or proteolytic fragment or recombinant or conjugated derivative of claim 1 or 2 or the fragment of claim 3 or any derivative of claim 4-8 with a second tenascin-specific antibody A composition comprising 32. Use of the composition of claim 30 for measuring circulating tenascin levels in a sandwich in vitro ELISA assay under conditions in which the second antibody binds to a second antigenic epitope. 32. Use of claim 31 for measuring the level of an isoform comprising an A _(1-4) -D region.

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